Method for isolating total RNA from cells

Abstract

Provided herein are methods for isolating cellular ribonucleic acid (RNA) from cells. The method includes suspending cells in an extraction solution comprising formamide; incubating the cells and formamide mixture; and pelleting cell debris, DNA, and protein to form an RNA-containing supernatant. Also provided herein are kits for isolating RNA and solutions for extracting RNA from a cell.

What is claimed is: 
     
       1. A method for isolating cellular ribonucleic acid (RNA), comprising:
 (a) suspending cells in an extraction solution comprising formamide in an amount of at least 40% by volume of the extraction solution; 
 (b) incubating the cells and formamide mixture of step (a); and 
 (c) pelleting cell debris, DNA, and protein to form an RNA-containing supernatant. 
 
     
     
       2. The method of  claim 1 , further comprising adding zirconium beads prior to incubating the mixture. 
     
     
       3. The method of  claim 1 , further comprising pelleting the cells prior to suspending the cells in the extraction solution. 
     
     
       4. The method of  claim 1 , wherein the extraction solution further comprises a reducing agent, an anionic detergent, a chelating agent, or a mixture thereof. 
     
     
       5. The method of  claim 4 , wherein the reducing agent comprises β-mercaptoethanol or dithiothreitol. 
     
     
       6. The method of  claim 5 , wherein the β-mercaptoethanol is present in the extraction solution in an amount of less than 5% by volume of the extraction solution. 
     
     
       7. The method of  claim 5 , wherein the β-mercaptoethanol is present in the extraction solution in an amount of less than 2% by volume of the extraction solution. 
     
     
       8. The method of  claim 5 , wherein the dithiothreitol is present in the extraction solution at a concentration of from 50 μM to 1 M. 
     
     
       9. The method of  claim 4 , wherein the anionic detergent comprises sodium dodecyl sulfate. 
     
     
       10. The method of  claim 4 , wherein the anionic detergent is present in the extraction solution in an amount of less than 5% by weight/volume of the extraction solution. 
     
     
       11. The method of  claim 4 , wherein the anionic detergent is present in the extraction solution in an amount of less than 0.05% by weight/volume of the extraction solution. 
     
     
       12. The method of  claim 4 , wherein the chelating agent comprises EDTA. 
     
     
       13. The method of  claim 4 , wherein the chelating agent is present in the extraction solution at a concentration of from 0.5 mM to 50 mM. 
     
     
       14. The method of  claim 4 , wherein the chelating agent is present in the extraction solution at a concentration of 18 mM. 
     
     
       15. The method of  claim 1 , wherein the extraction solution is substantially free from quaternary amine surfactants. 
     
     
       16. The method of  claim 1 , wherein the incubating step is performed at a temperature of from 10° C. to 140° C. 
     
     
       17. The method of  claim 1 , wherein the incubating step is performed at a temperature of 95° C.